The Transient Receptor Potential Vanilloid 1 channel (TRPV1) is a key component of nociceptors, a mechanosensor in the urinary bladder, and an osmosensor in neurons. The water permeability channels aquaporin-0 (AQP-0) and aquaporin-4 (AQP-4) are important for water homeostasis in the lens and brain, respectively. Although all of these channels are critical to normal body function and several pathological conditions, little is currently known about how their functions are modified by bilayer composition or their membrane microdomain locations. These are fundamental issues because some channels are sequestered into cholesterol-rich plasma membrane microdomains called rafts, and the functions of some ion channels are modified by membrane material properties controlled by cholesterol, as well as by specific interactions between cholesterol and the channel. Our preliminary studies show that TRPV1 activity depends strongly on membrane cholesterol content. This application tests the following two hypotheses: (1) key factors in sorting channels between microdomains are specific protein-lipid and protein- protein interactions, including homo-oligomerization of the channel, and (2) bilayer material properties (as controlled by phospholipid hydrocarbon chain composition and cholesterol content) modulate the functions of TRPV1, AQP-0, and AQP-4 channels. To determine the microdomain locations of TRPV1, AQP- 0, and AQP-4 we will employ a variety of techniques, including confocal microscopy and freeze-fracture electron microscopy. The functions of these channels in different lipid environments will be determined using patch clamp electrophysiology for TRPV1 currents and micropipette aspiration for AQP water permeability. The results of these experiments are expected to provide fundamental information on protein-lipid interactions and membrane microdomain formation, as well as further our understanding of nociception and the formation of lens cataracts.